The Influence of Microwave-Assisted Extraction on the Phenolic Compound Profile and Biological Activities of Extracts from Selected Scutellaria Species

The aim of the study was to investigate the effects of microwave-assisted extraction (MAE) conditions (microwave power, extraction time, and ethanol concentration) on the efficiency of the extraction of phenolic compounds from selected plant species belonging to the genus Scutellaria (i.e., Scutellaria baicalensis and Scutellaria lateriflora). The extracts from selected Scutellaria species were examined to establish the total phenolic content and the in vitro antioxidant and anti-inflammatory activity. The antioxidant capacity was determined by the ferric reducing antioxidant power (FRAP) and 2,2,1-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity methods. The anti-inflammatory activity was evaluated through the lipoxygenase (LOX) inhibitory assay. The phenolic profile of the extracts was characterized using ultra-high performance liquid chromatography coupled with diode array detection and high-resolution electrospray ionization mass spectrometry (UHPLC–DAD/ESI–HRMS/MS). Depending on the type of solvent and the extraction conditions used, the extracts obtained from selected Scutellaria species showed different total and individual phenolic content, as well as different antioxidant and anti-inflammatory properties. The results showed that all Scutellaria extracts had high total phenolic content and exhibited strong ferric ion reducing power and free radical scavenging capacity and a significant ability to inhibit the LOX activity. In general, the 70% ethanol extracts contained more phenolic compounds, mainly flavones, flavanones, and their derivatives, and showed greater in vitro biological activity than other extracts. The highest levels of phenolic compounds and the strongest antioxidant and anti-inflammatory potential were found in extracts from the roots of S. baicalensis. Optimal extraction conditions for all the plant materials tested were determined as the microwave power of 63 W, extraction time of 10 min, and 70% ethanol as the solvent.


Introduction
Plants of the genus Scutellaria belonging to the Lamiaceae family are cosmopolitan and are present across different continents with different climatic zones, mainly in North America, Europe, and East Asia. Species belonging to the genus Scutellaria L. are annual or perennial herbaceous plants [1][2][3]. Scutellaria baicalensis Georgi (known as Chinese Skullcap is converted into heat by ionic conduction or dipole rotation, so that heat is generated directly in the plant tissue. In addition, microwave heating creates significant pressure within the plant matrix. This improves the porosity of the cell structure, allowing the extraction solvent to penetrate more easily and facilitating extraction by various methods using water or ethanol for the solvent diffusion of compounds from the plant matrix [38]. As a result, MAE allows for effective extraction in a very short time, using only a small amount of solvent [29][30][31][32][33][34][35][36][37][38]. However, for high-quality extracts, not only the extraction method but also its parameters are very important [33,[37][38][39][40]. Generally, organic solvents (methanol and ethanol), water, and their mixtures have been widely used to isolate phenolic compounds, including flavonoids, from various plants using MAE. The efficiency of the MAE process depends mainly on the microwave power and the type of solvent, which must be a polar compound such as water, methanol, ethanol, and their mixtures [37]. According to Ni et al. (2018) [39], the solid-liquid ratio also plays a crucial role in attaining the maximum extraction yield of baicalin. Although previous studies have reported the effects of different extraction methods on the phytochemicals and bioactivity of S. baicalensis root extracts, no studies have determined the effects of different MAE conditions (the type of solvent, irradiation time, microwave power) on the total phenolic content, antioxidant activity, and inhibitory effect on lipoxygenase activity, as well as the extraction efficiency of phenolic compounds from the aerial parts of S. baicalensis and S. lateriflora.
Therefore, the aim of this study was to evaluate and optimize the MAE process of phenolic compounds (mainly flavonoids) from selected species belonging to the genus Scutellaria (i.e., S. baicalensis and S. lateriflora), and to investigate the qualitative and quantitative compositions of the phenolic compounds and the bioactivity (antioxidant and antiinflammatory) of the obtained extracts. The optimization of the extraction process included the selection of the extraction solvent and the determination of the effect of the time and the power of the microwave radiation on the extraction efficiency of phenolic compounds and the antioxidant properties of the obtained extracts. Different microwave powers (21,42, and 63 W) and extraction times (5 and 10 min) and three extractants (water, 40% ethanol, and 70% ethanol) in different combinations were used. The total phenolic content of the extracts was determined using the Folin-Ciocalteu assay. The antioxidant activity was evaluated using different in vitro tests, including 2,2,1-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and ferric reducing antioxidant power (FRAP). The in vitro anti-inflammatory activity of the extracts was determined by investigating the inhibition of the lipoxygenase (LOX) enzyme. The profile of phenolic compounds in extracts of dried roots and leaves of S. baicalensis and dried leaves of S. lateriflora was determined using ultra-high performance liquid chromatography coupled with diode array detection and high-resolution electrospray ionization mass spectrometry (UHPLC-DAD/ESI-HRMS/MS).

The Extraction Yield and Total Phenolic Content of the Extracts of Selected Species of Scutellaria
This is the first study that has attempted to determine the effects of different MAE conditions on the profile of phenolic compounds and the bioactivity of extracts from the roots and leaves of S. baicalensis and leaves of S. lateriflora. In order to evaluate the efficiency and optimize the extraction of phenolic compounds from the underground and aerial parts of selected Scutellaria spp. Using MAE, different solvents (water, 40 and 70% ethanol), microwave powers (21,42, and 63 W), and extraction times (5 and 10 min) were used. Water is a widely used extraction solvent because it is non-toxic, non-flammable, readily available, and environmentally friendly, and its physicochemical properties change with temperature and pressure [41]. However, several studies have shown that methanol and ethanol are the most efficient organic solvents for the extraction of flavonoids from Scutellaria spp. plants [42]. Since methanol is highly toxic and not suitable for food and pharmaceutical processing, ethanol is much more commonly used for the extraction of bioactive compounds, such as flavonoids, from plant materials because of its effectiveness and safety [38]. Ethanol and ethanol-water solutions are less toxic and more sustainable than other organic solvents, but they can also have harmful biological effects on the human body. However, ethanol can be easily recovered by distillation under reduced pressure. Previous studies have also shown that the extraction efficiency of flavonoids from the roots and leaves of plants belonging to Scutellaria spp. can be improved by replacing water and pure ethanol with a mixture of ethanol with water at concentrations of 40-50% (v/v) [32] and 60-70% (v/v) [43]. Thus, in the current study, water and 40 and 70% ethanol were used as extraction solvents. The extraction yield of phenolic compounds from plant materials is also significantly influenced by the solid to solvent ratio [39]. Generally, a higher solvent/solid ratio is required to achieve a higher extraction yield of phenolic compounds. However, if the solvent to solid ratio is too high, excessive extraction solvent is consumed, requiring longer extraction times [39]. Therefore, based on previous studies [44], a 1:20 (w/v) ratio of dried plant tissue to solvent was chosen.
The influence of the extraction parameters, such as the microwave power, time of irradiation, and solvent, on the extraction yield are shown in Table 1. The results showed that the MAE conditions had significant effects on the extraction yields of phenolic compounds from different Scutellaria spp. plant materials. The crude extract yield ranged from 249.36 mg/g to 373.47 mg/g for dried leaves of S. baicalensis, from 279.77 mg/g to 386.05 mg/g for dried leaves of S. lateriflora, and from 349.16 to 488.03 mg/g for dried roots of S. baicalensis. Regardless of the plant material, the highest yields were observed for extracts obtained at the highest microwave power and a longer extraction time. It was found that increasing the ethanol concentration up to 70% increased the extraction yield. These results are consistent with other reported results [30,33,43,45]. Other authors also showed that the extraction of S. baicalensis roots using water with ethanol concentrations of 60-70% (v/v) allows higher yields of phenolic compounds to be obtained than when using water or ethanol as a solvent [43]. In the present study, the optimal MAE conditions for the maximum crude extract yields from different Scutellaria spp. were microwave power of 63 W, an irradiation time of 10 min, and 70% ethanol as a solvent. Under these conditions, the yields of the crude extracts from the roots and leaves of Scutellaria spp. were considerably higher than those obtained by other authors [33,43].
The total phenolic content values of the extracts of the dried roots and leaves of S. baicalensis and the dried leaves of S. lateriflora are shown in Figure 1. The values obtained for total phenolic content are expressed as mg baicalin per g dry weight (mg BE/g DW). The total phenolic content values of the extracts of the dried roots and leaves of S. baicalensis and the dried leaves of S. lateriflora are shown in Figure 1. The values obtained for total phenolic content are expressed as mg baicalin per g dry weight (mg BE/g DW).  Results are presented as mean ± standard deviation of three replicates (n = 3). Bars with the same superscript (a-k) are not significantly different from each other (Tukey's HSD test, p < 0.05).
The results showed that regardless of the plant material and extraction conditions (microwave power and extraction time), the use of water as a solvent resulted in extracts with the lowest total content of phenolic compounds compared to ethanolic extracts. The extraction of phenolic compounds from the aerial and underground parts of selected Scutellaria plants was facilitated by the use of 40 and 70% ethanol as an extractant. The highest amount of phenolics was extracted from both the roots and leaves of S. baicalensis and S. lateriflora using 70% ethanol. Higher concentrations of ethanol were not used in this study because our previous experiments [44] have shown that the use of higher concentrations of ethanol (above 70%) results in lower extraction yields and lower total phenolic content. This phenomenon may be due to the denaturation of plant tissue proteins, which blocks the release of bioactive compounds into the solution [39]. Other authors compared the efficiency of S. baicalensis root and S. lateriflora aerial part extraction with various methods using water or ethanol as solvents [33,46]. Zhou et al. (2016) [33] showed that the most effective method for the extraction of phenolic compounds from S. baicalensis roots was MAE using water as a solvent. They also revealed that ultrasonic-assisted extraction (UAE) with 70% ethanol produced higher yields of phenolic compounds than UAE with water [33]. Similarly, Lohan et al. (2013) showed that ethanolic extracts of S. lateriflora contain more phenolic compounds than water ones [46].
The total phenolic content of the extracts of the different Scutellaria species was also significantly affected by the microwave power and extraction time. The total phenolic content of almost all extracts obtained from selected Scutellaria plants was significantly increased (p < 0.05) upon increasing the microwave power from 21 to 63 W. The exceptions were the ethanolic extracts of the dried roots of S. baicalensis and all extracts of the dried leaves of the same species. Increasing the microwave power to 63 W decreased the total phenolic content of these extracts. The microwave power of 42 W was found to be the optimal value in this case. Higher microwave power did not increase the content of phenolic compounds in ethanolic extracts but increased the total phenolic content in water extracts. This may indicate that some of the extracted compounds were degraded by exposure to higher microwave powers.
Regardless of the solvent and microwave power, a longer extraction time usually resulted in higher phenolic content in the extracts. The exception was the extraction of phenolic compounds from both parts of S. baicalensis using 40 and 70% ethanol as a solvent and the highest microwave power (63 W). In this case, a longer extraction time resulted in lower content of phenolic compounds. This may indicate that overly long exposure to microwaves leads to the degradation or transformation of the extracted compounds. However, for the dried leaves of S. lateriflora, increasing the extraction time and microwave power resulted in an increase in the total phenolic content. These differences in total phenolic content in all extracts analyzed can be attributed to differences in their composition.
The total phenolic content of extracts obtained from leaves of both S. baicalensis and S. lateriflora was significantly lower than that of S. baicalensis root extracts. The highest total phenolic content of extracts was obtained from the dried roots of S. baicalensis (115.93 mg BE/g DW) when 70% ethanol was used as a solvent and the microwave power and extraction time were 42 W and 10 min, respectively. The water extract obtained from the leaves of S. baicalensis during 5 min extraction at 21 W was characterized by the lowest total phenolic content (38.37 mg BE/g).
According to these results, the optimal extraction conditions for obtaining the highest total phenolic content from both roots and leaves of S. baicalensis were found to be 70% ethanol, an extraction time of 10 min, and microwave power of 42 W. Meanwhile, in the case of the leaves of S. lateriflora, the optimal MAE conditions were 70% ethanol, 10 min, and 63 W.

Antioxidant and Anti-Inflammatory Activity of the Extracts of Selected Species of Scutellaria
The results showed that the conditions of the MAE process and the type of extractant have a significant effect on the antioxidant and anti-inflammatory properties of extracts

Antioxidant and Anti-Inflammatory Activity of the Extracts of Selected Species of Scutellaria
The results showed that the conditions of the MAE process and the type of extractant have a significant effect on the antioxidant and anti-inflammatory properties of extracts from selected plant materials of the genus Scutellaria. Depending on the type of solvent and the extraction conditions used, the extracts obtained showed different antioxidant activity, measured using various in vitro tests (Figures 2 and 3).  Bars with the same superscript (a-l) are not significantly different from each other (Tukey's HSD test, p < 0.05). In general, S. baicalensis root extracts were characterized by higher ferric reducing activity when compared to extracts obtained from the leaves of both Scutellaria species ( Figure 2). It was also found that the ethanolic extracts of selected Scutellaria plant organs In general, S. baicalensis root extracts were characterized by higher ferric reducing activity when compared to extracts obtained from the leaves of both Scutellaria species ( Figure 2). It was also found that the ethanolic extracts of selected Scutellaria plant organs had higher FRAP values than the water extracts. The ferric ion reduction ability of almost all extracts was increased upon increasing the microwave power from 21 to 63 W. Similarly, in most cases, an increase in extraction time contributed to an increase in FRAP values. The exceptions were the extracts of the dried leaves of S. baicalensis and water extracts of the dried leaves of S. lateriflora. The highest antioxidant capacity measured by the FRAP assay was noticed for 40% ethanolic extracts obtained from dried roots of S. baicalensis using microwave power of 63 W in 10 min (528.37 µM TE/g DW). The extract obtained from the leaves of S. baicalensis showed the highest activity compared to all other extracts from this material when 70% ethanol was used as a solvent and extraction was carried out for 10 min at 42 W. Similarly to the total phenolic content, the extract obtained from the leaves S. baicalensis after 5 min extraction at 21 W microwave power had the lowest ferric ion reduction capacity (164.35 µM TE/g DW).
The results of the present study showed also that the free radical scavenging capacity tended to increase when extending the extraction time in the case of extracts obtained from the roots and leaves of the studied Scutellaria species (Figure 3). Increasing the microwave power to 63 W increased the DPPH radical scavenging capacity of the extract compared to the lower microwave powers. The exception was the water extracts that were obtained from the dried leaves of S. baicalensis. In the case of the extracts obtained from the leaves of the two Scutellaria species tested, the ethanolic extracts showed a significantly greater ability to scavenge DPPH free radicals than the water extracts, irrespective of the extraction time and microwave power used. In contrast, in the case of S. baicalensis roots, water extracts obtained using lower microwave powers (21 and 42 W) showed higher free radical scavenging activity than ethanolic extracts. Nonetheless, of all the extracts tested, those obtained from S. baicalensis roots using 40% ethanol, an extraction time of 10 min, and a microwave power of 63 W had the highest ability to scavenge DPPH free radicals (414.57 µM TE/g DW). For the extract obtained from the leaves of S. baicalensis using water as a solvent, the shorter extraction time (5 min) and the highest microwave power (63 W) resulted in the lowest capacity for scavenging free radicals (165.87 µM TE/g DW).
The results of the DPPH radical experiments were also expressed as IC 50 values to compare the antioxidant activity of extracts from roots and leaves of the studied Scutellaria species obtained under optimal MAE conditions with the results of previous studies. Under optimal conditions, the IC 50 values of extracts from dried roots and leaves of S. baicalensis and dried leaves of S. lateriflora were found to be 38.64, 43.41, and 37.46 µg/mL, respectively (Table S1). Interestingly, the IC 50 values obtained in the current study are significantly lower than those reported by other authors for extracts of the roots of S. baicalensis [33] and aerial parts of S. lateriflora [46]. Since a low IC 50 value indicates strong DPPH radical scavenging activity, the results obtained confirm that the optimized extraction method yields extracts with very strong anti-free-radical properties.
In the present study, the in vitro anti-inflammatory activity of extracts obtained from the aerial and underground parts of selected Scutellaria spp. was also tested by evaluating their potential to inhibit the activity of lipoxygenase (LOX). Lipoxygenases are ironcontaining non-heme dioxygenases. These enzymes are responsible for the formation of hydroperoxides from polyunsaturated fatty acids such as linoleic acid and arachidonic acid and for the oxidative modification of low-density lipoproteins (LDL). LOX is also involved in the progression of inflammatory processes [47,48]. During the early phase of inflammation, arachidonic acid is predominantly metabolized by 5-lipoxygenase (5-LOX), which generates a variety of mediator molecules, including highly pro-inflammatory leukotrienes such as leukotriene (LT) B4, LTC4, and LTD4 [48]. Therefore, bioactive compounds acting as LOX inhibitors are of great interest in controlling inflammatory processes [47]. The results of LOX inhibition for the extracts of the dried roots and leaves of S. baicalensis and the dried leaves of S. lateriflora are shown in Table 2. It was observed that all the extracts could inhibit the activity of this enzyme. However, the extracts obtained showed variable LOX inhibitory activity depending on the plant material, the type of solvent, and the extraction conditions used. The extracts from the roots of S. baicalensis displayed stronger inhibitory activity towards LOX (IC 50 , 28.16-44.12 µg/mL) than the extracts from the leaves of the same Scutellaria spp. and S. lateriflora, with IC 50 values in the range of 56.35-77.30 µg/mL and 39.31-61.87 µg/mL, respectively. The greatest inhibition of this enzyme was observed for the extract from the dried roots of S. baicalensis obtained at the microwave power of 63 W and the irradiation time of 10 min with 70% ethanol as the solvent. The lowest LOX inhibitory activity was exhibited by the water extract obtained from the leaves of the same Scutellaria spp. exposed to microwave power of 21 W for 5 min. LOX inhibition of the aerial and underground parts of selected Scutellaria spp. extracts was similar or higher than the extracts for other common plants [49]. However, it was observed that all extracts possessed significantly lower LOX inhibitory activity than pure standards (baicalin and baicalein). Nonetheless, the results of this study suggest that the extracts from both Scutellaria spp. plant materials, due to the presence of a high amount of bioactive compounds, were effective in inhibiting LOX activity and could reduce the production of pro-inflammatory molecules.
The leaves and roots of different Scutellaria spp. plants are composed of different phenolic compounds with different polarities [36][37][38][39][40][41][42]. Based on the result of the total phenolic content, antioxidant capacity, and inhibition of lipoxygenase activity of the obtained extracts, it was suggested that most of the bioactive compounds present in both S. baicalensis and S. lateriflora leaves and S. baicalensis roots had a moderately polar nature. Thus, the content of these compounds in the extracts increased when the ethanol concentration was increased to 70%.

Phenolic Profiles of the Extracts of Selected Species of Scutellaria
The characterization of the phenolic profiles of the roots and leaves of S. baicalensis and leaves of S. lateriflora extracts was performed using UHPLC-DAD/ESI-HRMS/MS. Table 3 shows the phenolic compounds tentatively identified on the basis of absorption maxima in UV-vis spectra, full-scan mass spectra, MS/MS fragmentation patterns, retention times, and chromatographic elution order, and a comparison with standard compounds and literature data [50][51][52][53][54][55]. The typical chromatograms of the extracts of selected plant materials belonging to the genus Scutellaria are shown in Figure 4, and the structures of the identified phenolic compounds are shown in Figure S1.
In this study, a total of 16 phenolic compounds were identified in the roots of S. baicalensis, a total of 15 in the leaves of S. baicalensis, and 15 in the leaves of S. lateriflora, all of which were flavonoids, mainly flavones, flavanones, and their derivatives.
Both qualitative and quantitative differences in the composition of phenolic compounds were observed among the different species of Scutellaria and the different MAE parameters (Tables 4-6). The total amount of identified phenolic compounds, calculated as the sum of individual compounds, in the obtained extracts ranged from 1.03 to 102.75 mg/g DW. Results are presented as the mean ±SD from triplicate determinations; nd-not detected; Chry-Ara-Glu-chrysin 6-C-arabinoside-8-C-glucoside; Chry-Glu-Ara-chrysin 6-C-glucoside-8-    Results are presented as the mean ±SD from triplicate determinations; nd-not detected; IsoCart-GluA-isocarthamidin 7-O-glucuronide; Chry-Ara-Glu-chrysin 6-C-arabinoside-8- The ethanolic extracts obtained from the aerial and underground parts of the selected Scutellaria species showed significantly (p < 0.05) higher content of phenolic compounds than those obtained with water as the solvent, regardless of the extraction conditions used. In addition, the total amount of phenolic compounds in the extracts of all plant materials tested increased when the ethanol concentration was increased from 40 to 70%. It was also confirmed by other authors that 70% ethanol is the best solvent for the extraction of the flavonoids from the different species of the genus Scutellaria [40]. However, Zu et al. (2016) [33] demonstrated that microwave-assisted extraction using water at 300 W at 100 • C for 2 h was the most efficient method for the extraction of phenolic compounds from S. baicalensis roots. Bergeron et al. (2005) [41] showed that extraction with either water at 85 • C for 30 min or with 70% ethanol for 24 h at room temperature yields the highest amounts of flavonoids present in the leaves of S. lateriflora. Besides the fact that the solubility of the majority of substances usually increases with temperature, it is also believed that a high temperature reduces the polarity of water and increases its ability to dissolve non-polar compounds such as baicalin, baicalein, scutellarin, lateriflorin, dihydrobaicalin, and oroxylin A 7-O-glucuronide [41]. Due to increased solvent diffusion into the plant matrix and the increased solubility and desorption of phenolic compounds from the matrix, higher temperatures result in higher extraction yields. However, when higher temperatures are used, the degradation of heatsensitive compounds may occur [55]. In the present study, the extraction temperature did not exceed 60 • C. Therefore, water was the least effective solvent for the flavonoids present in Scutellaria roots and leaves.
The concentration of phenolic compounds in the extracts of the different Scutellaria species was also considerably affected by the extraction time and microwave power (Tables 4-6). It was also observed that the extraction efficiency of phenolic compounds increased as the extraction time increased from 5 to 10 min. The total phenolic compound content of almost all extracts obtained was also increased upon increasing the microwave power from 21 to 63 W.
Furthermore, it was found that ethanolic extracts obtained from the roots of S. baicalensis had significantly higher content of total phenolic compounds (81.15-102.75 mg/g DW) than those obtained from the leaves of both species (30.75-51.95 mg/g DW), regardless of the ethanol concentration and extraction conditions used. The highest total quantity of the selected individual phenolic compounds was observed for the extract obtained from the roots of S. baicalensis with 70% ethanol for 10 min at the microwave power of 63 W, while the lowest concentration of phenolic compounds was found in the water extract obtained from the leaves of S. lateriflora during 5 min extraction at 21 W.
Baicalin (31.14-36.33 mg/g DW) was detected in the highest concentration in the ethanolic extracts of the leaves of S. lateriflora, followed by dihydrobaicalin (3.30-4.38 mg/g DW).
The levels of oroxyloside, hydroxyldimethoxyflavanone glucuronide, wogonoside hispiduloside, baicalein, and scutellarin in the ethanolic extracts of these leaves depended on the ethanol concentration and extraction conditions and fluctuated within the range of 1.69-2.04, 1.57-1.93, 1.53-1.90, 1.53-1.84, 0.48-1.61, and 0.96-1.16 mg/g DW, respectively. The other phenolic compounds were found in much lower concentrations in S. lateriflora ethanolic extracts (0.01-0.76 mg/g DW). The total content of the investigated phenolic compounds in the ethanolic extracts of these leaves ranged from 44.60 to 51.95 mg/g DW. The differences in the profiles and levels of phenolic compounds between the different morphological parts of the Scutellaria plants were due to the features of the different species [50][51][52][53][54].
Interestingly, the water extracts from the leaves of S. baicalensis were characterized by a significantly higher total concentration of phenolic compounds (5.94-7.91 mg/g DW), compared to the water extracts from the other aerial and underground parts of the studied Scutellaria species (1.03-4.36 mg/g DW). The leaves of S. baicalensis differed from the roots of the same species and the leaves of S. lateriflora by the presence of high amounts of tetrahydroxyflavanone and tetrahydroxyflavone glucuronides, mainly isocarthamidin 7-O-glucuronide and scutellarin, structurally related to (S)-naringenin and apigenin, respectively. These compounds are more hydrophilic than baicalin, baicalein, and dihydrobaicalin, the major phenolic compounds present in S. baicalensis roots and S. lateriflora leaves, and can be efficiently extracted even when water is used as a solvent. Thus, the two predominant compounds in water extracts of S. baicalensis were isocarthamidin 7-O-glucuronide (1.39-1.95 mg/g DW) and scutellarin (1.39-1.91 mg/g DW), and these were followed by api- In contrast, much fewer phenolic compounds were detected in the water extracts of the roots of S. baicalensis and leaves of S. lateriflora (Tables 4-6). The water extract of S. baicalensis roots was dominated by chrysin 6-C-arabinoside-8-C-glucoside (0.99-1.75 mg/g DW) and chrysin 6-C-glucoside-8-C-arabinoside (0.66-1.18 mg/g DW). Interestingly, the content of these two compounds was only slightly lower compared to their content in ethanolic extracts. Meanwhile, the content of baicalin (0.46-1.22 mg/g DW) and baicalein (0.04-0.13 mg/g DW) in water extracts of this raw material was more than 40-fold and 135-fold lower, respectively, compared to ethanolic extracts. The other compounds detected were present only in trace amounts. These observations agree with the findings reported by Zhou et al. (2016) [33]. They also showed that when water was used as a solvent in the ultrasound-assisted extraction of S. baicalensis roots, the obtained extracts had significantly lower amounts of baicalin, baicalein, wogonoside, and wogonin than the UAE extracts obtained using 70% ethanol. However, the amount of water-soluble components such as chrysin 6-C-arabinoside-8-C-glucoside and chrysin 6-C-glucoside-8-C-arabinoside was only slightly lower [33].
Similar trends were observed for the water and ethanolic extracts of the leaves of S. lateriflora. Almost all phenolic compounds detected in water extracts of this plant material, especially baicalin, dihydrobaicalin, and baicalein, were found in lower concentrations than in ethanolic extracts. For the water extract of S. lateriflora leaves, the lowest recovery compared to the ethanolic extract was found for dihydrobaicalin. This compound was the second most abundant phenolic in the ethanolic extracts of this material, similar to the findings reported by Bergeron et al. (2005) [41]. In the present study, only trace amounts of dihydrobaicalin (0.02-0.03 mg/g DW) were found in the water extract, and only when the highest microwave power (63 W) was used during extraction. Other authors have also shown that water extracts of S. lateriflora leaves contain less dihydrobaicalin than ethanolic extracts [41].
It is not surprising that the total concentration of phenolic compounds determined by the UHPLC-DAD method differs from that obtained by the Folin-Ciocalteu (F-C) assay ( Figure 1 and Tables 4-6). The F-C method is often used to determine the total phenolic content in plant extracts. However, this assay is not specific to phenolic compounds only. Other non-phenolic-reducing agents such as ascorbic acid, sugars, amino acids, peptides, organic acids, aromatic amines, and Maillard reaction products may react with the F-C reagent and interfere with the determination results. The F-C test can be used to determine not only phenolic compounds but also other substances with reducing capacity [44,56]. Therefore, the results of the phenolic compounds determined by the F-C test and the UHPLC-DAD method may differ significantly. These differences may be mainly due to differences in the content of individual phenolic compounds in the obtained extracts, but they may also result from the transfer of other compounds present in the tested plant material to the extract, such as polysaccharides and their degradation products formed during microwave extraction [57].
In conclusion, the results obtained show that extraction with 70% ethanol at 10 min extraction time and microwave power of 63 W are the optimal MAE processing conditions for obtaining the highest content of most of the compounds analyzed, especially baicalin, baiclalein, wogonoside, wogonin, and isocarthamidin 7-O-glucuronide, from the roots and leaves of S. baicalensis and leaves of S. lateriflora.
The results of this study confirm the strong antioxidant properties of S. baicalensis root and S. lateriflora leaf extracts obtained by microwave extraction, suggesting their beneficial potential in the prevention of diseases associated with oxidative stress, such as cardiovascular and inflammatory diseases. The flavonoids contained in these extracts, especially baicalein and its glucuronides, may be responsible for their high antioxidant activity.
In vitro studies confirmed that baicalein can scavenge hydrogen peroxide, superoxide, and even hydroxyl radicals. These findings were confirmed in an ischemia-reperfusion model where the treatment of cardiomyocytes with S. baicalensis root extract reduced cell death from almost 50 to 23% [58]. The protective effect has also been confirmed in experiments using extracts from two other species of Scutellaria. Extracts from S. altissima and S. alpina have been proven to protect against oxidative damage to human plasma proteins and lipids [59], as well as blood platelets and lymphocytes [60]. Previous studies have shown that the major bioactive compounds found in Scutellaria spp., including baicalein, baicalin, wogonin, wogonoside, and oroxylin A, can inhibit the production of several pro-inflammatory cytokines and chemokines and pro-inflammatory mediators, while promoting the production of certain anti-inflammatory cytokines [25,26,61].

Plant Materials
The dried roots of S. baicalensis were purchased from Nanga (Blękwit, Poland) and dried leaves of S. baicalensis and S. lateriflora were obtained from Plantago (Złotów, Poland). All the samples were dried plant materials. Before the extraction, the plant materials were ground to powder (<0.50 mm) using a laboratory knife mill (GRINDOMIX GM 200, Retsch, Haan, Germany).

Microwave-Assisted Extraction (MAE)
The leaf and root powders were extracted in Teflon vessels using an RM-800 microwave reactor (Plazmatronika, Poland, Wroclaw) at a temperature not exceeding 60 • C, with continuous stirring using a built-in magnetic stirrer. The temperature during extraction was automatically and continuously measured and controlled by a Reflex fiber-optic temperature meter (Neoptix, Canada, Québec). MAE was performed using three different solvents (water, EtOH/H 2 O 40:60 v/v, and EtOH/H 2 O 70:30 v/v). The MAE parameters were the microwave power (21,42, and 63 W) and extraction time (5 and 10 min). The ratio of dry plant material to solvent used was 1:20 (w/v). Each extraction experiment was performed in triplicate. We selected the extraction parameters based on literature data [41] and our previous experience with other plants [44], as data on the use of microwaves to extract phenolic compounds from Scutellaria plants are still limited. After MAE, the extract was centrifuged at 1050× g for 10 min at 20 • C in the centrifuge type MPW-350 (MPW MED, Instruments, Warsaw, Poland). The supernatants were filtered through Whatman No. 4 filter paper and stored at −80 • C until further use. To determine the extraction yield, 10 mL of each supernatant was transferred to a pre-weighed 40 mL glass vial. Supernatants were evaporated to dryness at 40 • C.
The extraction yield was calculated using the following equation: Extraction yield (mg/g) = (X 1 /X 2 ) where X 1 is the weight (mg) of the dried extract and X 2 is the weight (g) of the plant material subjected to the extraction process.

Identification and Quantification of Polyphenolic Compounds by UHPLC-DAD/ESI-HRMS/MS
UHPLC-DAD and UHPLC-ESI-HRMS/MS analyses of polyphenolic compounds in Scutellaria plant extracts were carried out using a UHPLC+ Dionex UltiMate 3000 liquid chromatographic system (Thermo Fisher Scientific Inc., Waltham, MA, USA) equipped with a diode array detector and a Transcend™ TLX-2 multiplexed LC system coupled to a Q-Exactive hybrid quadrupole-orbitrap mass spectrometer with a heated electrospray ionization (HESI-II) source (Thermo Scientific, Hudson, NH, USA), respectively. Prior to analysis, the extracts were filtered through 0.20-µm nylon syringe filters. Chromatographic separation was performed on the Accucore™ C18 column (2.1 × 150 mm, 2.6 µm particle size; Thermo Scientific, PA, USA) in the gradient elution mode. The column was maintained at 30 • C and the flow rate was 0.35 mL/min. Eluent A was 0.1% formic acid in water and eluent B was acetonitrile. The gradient elution program was as follows: 0-8 min The injection volume was set to 10 µL. The chromatograms were recorded at 280 nm. The mass spectrometer was operated in negative ionization mode with full MS-SIM and a subsequent parallel reaction monitoring (PRM) mode. Full-MS-SIM and PRM spectra were obtained by scanning m/z from 100 to 1500. The mass spectrometer operation conditions were as follows: capillary temperature, 275 • C; heater gas temperature, 320 • C; electrospray capillary voltage, 4.5 kV; sheath and auxiliary gas, nitrogen; sheath gas flow, 35 arbitrary units; auxiliary gas flow, 15 arbitrary units; normalized collision energy (NCE), 20% [35]. The identification of polyphenolic compounds was performed by matching their retention times, spectra characteristics, full-scan mass spectra in negative ionization mode, and MS/MS fragmentation patterns with those of pure standards analyzed under identical conditions and literature data [50][51][52][53][54]. Quantification of individual polyphenolic compounds was carried out using the external standard method. All measurements were conducted in triplicate and results were expressed as mg per g DW (mg/g DW).

Determination of Total Phenolic Content
The total phenolic content of the extracts of selected Scutellaria plant materials was determined using the Folin-Ciocalteu method, as described by Oracz andŻyżelewicz (2019) [62]. The total polyphenolic content was calculated from the calibration curve prepared with the use of baicalin as the standard. Results of the total phenolic content were expressed as mg baicalin equivalents (BE) per gram of DW (mg BE/g DW).

Evaluation of the Antioxidant Activity
Two in vitro assays, namely free radical-scavenging capacity (DPPH) and ferric ion reducing antioxidant power (FRAP), were used to evaluate the antioxidant potential of the extracts of selected Scutellaria plant materials, as described by Oracz andŻyżelewicz (2019) [62]. The results of the DPPH and FRAP assays were expressed as µM Trolox equivalents per gram of DW (µM TE/g DW).

Evaluation of the Anti-Inflammatory Activity
In vitro inhibition of LOX activity was determined spectrophotometrically according to Michel et al. (2017) [63], with some minor modifications. Briefly, 50 µL of tested extract (50 to 500 µg/mL) was mixed with 950 µL of sodium borate buffer (pH = 9.0), 1 mL of LOX solution (300 U/mL), and 1 mL of linoleic acid solution (138 µM). The increase in absorbance of the reaction mixture was measured for each sample on a UV-1800 spectrophotometer (Shimadzu, Tokyo, Japan) at 234 nm over a period of 15 min. Baicalein and baicalin were used as reference standards, whereas sodium borate buffer solution was used as the control. The activity in the absence of the extract or standard was considered to be 100% of LOX activity.
The anti-inflammatory effect was evaluated by calculating the percentage inhibition of hydroperoxide production from the absorbance values at 234 nm after 10 min of incubation. The results of LOX inhibitory activity were expressed as the IC 50 value (µg/mL); a lower IC 50 value represents stronger anti-inflammatory activity.

Statistical Analysis
The results of three independent experiments were expressed as the mean ± standard deviation (SD). The Statistica 13.0 software (StatSoft, Inc., Tulsa, OK, USA) was used for the statistical data analyses. The significant differences were estimated by one-way analysis of variance (ANOVA) followed by Tukey's Honest Significant Difference (HSD) test. Differences were considered to be significant when p-values were less than 0.05 (p < 0.05).

Conclusions
This study reported the manipulation of the extraction conditions of the dried roots and leaves of S. baicalensis and dried leaves of S. lateriflora using the MAE method in order to obtain extracts with higher levels of phenolic compounds. The results of the research allowed the optimization of the microwave extraction process, including the choice of solvent, time, and microwave power, to maximize the total and individual phenolic compounds and the antioxidant and anti-inflammatory activity of extracts from the aerial and underground parts of selected Scutellaria plants. The optimal MAE processing conditions for obtaining the extracts from the roots and leaves of S. baicalensis and leaves of S. lateriflora, with the highest total and individual content of most phenolic compounds, especially baicalin, baicalein, wogonoside, wogonin, and isocarthamidin 7-O-glucuronide, and enhanced antioxidant and anti-inflammatory potential, were 70% ethanol as a solvent, an extraction time of 10 min, and microwave power of 63 W. It was found that under these conditions, MAE was a more efficient process than heat reflux and UAE for the extraction of bioactive compounds from the roots and leaves of selected Scutellaria species. In addition, the extracts obtained by the optimized extraction procedure showed significant antioxidant and anti-inflammatory activity and could be used as a natural source of bioactive compounds in the production of nutraceuticals and functional foods. The results of this study may be useful for the development of industrial MAE processes with improved extraction efficiency for selected bioactive compounds from different organs of S. baicalensis and S. lateriflora.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules28093877/s1. Table S1: The free radical scavenging activity DPPH assay as IC 50 of optimal extracts from dried roots and leaves of S. baicalensis and dried leaves of S. lateriflora. Figure S1: Structures of phenolic compounds found in extracts from dried roots and leaves of S. baicalensis and dried leaves of S. lateriflora. Chemical structures were drawn using ACD/ChemSketch software.